The aim of this review was to evaluate the importance of the real-time PCR (qRT-PCR) as a technique for mRNA expression analysis in different tissues. Real-time PCR is widely used for quantification of mRNA levels and is a fundamental tool for basic research, molecular medicine and biotechnology. Genes of references are expressed in a wide variety of tissues and cells with minimal variations in their expression levels, and thus are used to normalize data of mRNA quantification. Software programs, such as geNorm, BestKeeper and NormFinder, have been developed to perform the normalization of data, which help to choose the most stable reference gene. Several genes, such as GAPDH, β-actin, β-tubulin, PGK, UBQ, RPL-19 and 18S rRNA have been suggested as standards in PCR studies, but these genes can have variation in their expression in different tissues, reinforcing the idea that there is no ideal reference gene.
The aim of this study was to evaluate the effects of different concentrations of BMP4 on activation, development and mRNA expression of GDF9, BMP15, PCNA, Bax and Bcl2 in cultured bovine follicles enclosed in ovarian tissues. Ovarian tissue fragments were cultured for 6 days in α-MEM+ alone or supplemented with different concentrations of BMP4 (10, 50 or 100 ng/ml). Classical histology was performed to analyze follicle growth and morphology, while real-time PCR was used to analyze mRNA levels in fresh and cultured tissues. After 6 days, the culture of ovarian tissue in α-MEM+ alone or supplemented with 10, 50 or 100 ng/ml BMP4 promoted follicular activation. The different concentrations of BMP4 maintained the percentage of normal follicles similar to results of the control. The presence of 100 ng/ml BMP-4 in culture medium increased oocyte and follicular diameters of primary and secondary follicles when compared with those follicles from uncultured control or cultured in α-MEM+ alone (P < 0.05). The tissues cultured in the presence of increasing concentrations of BMP4 had an increase in mRNA expression of the tested genes, but despite this the differences were not statistically significant. In conclusion, 100 ng/ml BMP4 promotes an increase in diameters of follicles and oocytes of primary and secondary follicles after 6 days of in vitro culture.
Introduction The chromatin landscape of mammalian cells underpins their transcriptional profiles, which then dictate fate and function. Mutations within chromatin remodelling genes have been implicated in early oesophageal adenocarcinoma (OAC) development, thus alterations in the chromatin landscape may pose an important molecular step in OAC development. OAC is often lethal, therefore genome-wide, basic research will provide foundations to develop new treatments and patient stratification methods. Material and methods We have used Assay for TransposaseAccessible Chromatin using sequencing (ATAC-seq) to profile the accessible chromatin landscape of normal oesophagus and oesophageal adenocarcinoma tissue samples, in addition to representative cell lines. Downstream analysis has involved bioinformatic de novo motif analysis and ATAC-footprinting, Chromatin immunoprecipitation with sequencing (ChIP-seq) and siRNA-mediated knockdowns. Results and discussions Our results revealed an altered chromatin landscape in cancerous tissue and identified 'cancer-specific' regions of accessibility. Using de novo motif discovery methods and footprinting analysis, we have identified enriched transcription factor motifs for GATA6 and HNF4A in differentially accessible regions and footprints. Also, comparison between the chromatin landscape of OAC cell-lines and OAC tissue identified the cell-line that best represents OAC tumours. HNF4A ChIP-seq and GATA6 ChIP-seq data, in oesophageal cancer cells, shows co-occupancy of HNF4A and GATA6 at 90% of sites and confirm motif enrichment and footprint observations. HNF4A and GATA6 knockdown experiments demonstrate overlap of gene regulation and also identify genes that are exclusively regulated by the presence HNF4A and GATA6. Importantly, genes directly regulated by HNF4A or GATA6 are on average overexpressed in OAC, however genes regulated by both factors are more overexpressed by 2-fold. Conclusion Our data demonstrate the power of ATAC-seq in genome-wide discovery methods and we have identified a novel HNF4A-GATA6 transcription network that is active in OAC.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.